ABSTRACT The inherited mechanobullous disease, dystrophic epidermolysis bullosa, is caused by type VII collagen gene (COL7A1) mutations. We studied six unrelated patients with a distinct clinical subtype of this disease, epidermolysis bullosa pruriginosa, characterized by pruritus, excoriated prurigo nodules, and skin fragility. Mutation analysis using polymerase chain reaction amplification of genomic DNA, heteroduplex analysis and direct nucleotide sequencing demonstrated pathogenetic COL7A1 mutations in each case. Four patients had a glycine substitution mutation on one COL7A1 allele (G1791E, G2242R, G2369S, and G2713R), a fifth was a compound heterozygote for a splice site mutation (5532 + 1G-to-A) and a single base pair deletion (7786delG), and a sixth patient was heterozygous for an out-of-frame deletion mutation (6863del16). This study shows that the molecular pathology in patients with the distinctive clinical features of epidermolysis bullosa pruriginosa is heterogeneous and suggests that other factors, in addition to the inherent COL7A1 mutation(s), may be responsible for an epidermolysis bullosa pruriginosa phenotype.Keywords: basement membrane zone, genetic blistering skin disease, pruritus

VOL. 112, NO. 6JUNE 1999COL7A1 MUTATIONS IN EPIDERMOLYSIS BULLOSA PRURIGINOSA985Table I. Mutation analysis reveals allelic heterogeneity forCOL7A1 mutations in epidermolysis bullosa pruriginosaPatientInheritancePositionMutationVerification12345DominantNot knownbNot knowncDominantRecessiveExon 85Exon 61Exon 93Exon 110Intron 64G2242RG1791EG2369SG2713R5532 ? 1G-to-A(de novo)7786delG6863del16SacIaBlpIHphIEco01091TaiIExon 104Exon 87BsaJIsequencing6DominantaSacI restriction digest using modified exon 85 primer (Lee et al, 1997).bMutation excluded from maternal DNA, but paternal DNA unavailable for study.cDNA from other family members not available for study.occasional foci of sublamina densa cleavage, and normal or near-normalexpression of type VII collagen in all three cases.Mutation analysislymphocytes of the six patients under study and family members whereavailable by standard methods (Sambrook et al, 1989), and used as atemplate for polymerase chain reaction (PCR) amplification of COL7A1using oligonucleotide primer pairs spanning all 118 exons of the gene andPCR conditions described previously (GenBank numbers L02870, L23982)(Christiano et al, 1997). In addition, a mismatch PCR method was usedto create an artificial SacI restriction site for screening exon 85 PCRproducts for the mutation G2242R as described by Lee et al, 1997. Fivemicroliter aliquots of PCR products were analyzed by 2% agarose gelelectrophoresis then 3–8 µl of products were prepared for heteroduplexanalysis using conformation-sensitive gel electrophoresis (Ganguly et al,1993).PCRproductsdemonstratingalteredelectrophoreticmobilityunder-went direct nucleotide sequencing in an ABI 310 genetic analyzer (PerkinElmer, Warrington, U.K.), and mutations were confirmed by restrictionendonuclease digestion, performed according to the manufacturer’s recom-mendations (New England BioLabs, Hitchin, U.K.). Mutations detectedwere excluded from 100 normal chromosomes using restriction enzymeanalysis or direct nucleotide sequencing.Genomic DNA was extracted from peripheral bloodRESULTS AND DISCUSSIONThe pathogenetic mutations in COL7A1 identified in the six casesare summarized in Table I.Initial PCR of exon 85 using modified primers to create anartificial restriction site for SacI (Lee et al, 1997) revealed thepresence of G2242R on one COL7A1 allele in patient 1 but notin any of the other EB pruriginosa patients. This G-to-A transition,which converts a glycine residue (GGG) to an arginine (AGG),was confirmed in the patient and his father by direct nucleotidesequencing.The mutationwas notdetected inthe patient’sclinicallyunaffected mother or daughter. In the remaining five patients,mutation analysis of COL7A1 identified different mutations in eachcase. Patients 2–4 had previously undescribed glycine substitutionmutations. The glycine substitutions in cases 2 and 3 were thoughtto be de novo dominant mutations, although a recessive mode ofinheritance with failure to detect the second mutation on hetero-duplex analysis could not be ruled out. In patient 2, the glycinesubstitution arose from a G-to-A transition at nucleotide position5372 in exon 61, converting a glycine residue (GGG) to glutamicacid (GAG), G1791E. This mutation was not present in DNAfrom the patient’s clinically unaffected mother, but paternal DNAwas not available for analysis. In patient 3, a G-to-A transition atnucleotide position 7105 in exon 93, converting a glycine (GGT)to serine (AGT), G2369S, was identified. DNA was not availablefrom parents or other family members to assess the inheritance ofthis mutation. In patient 4, a G-to-C transversion at nucleotideposition 8137 in exon 110 was delineated, resulting in the conver-sion of a glycine (GGC) to arginine (CGC), G2713R. This glycinesubstitution was also identified in patient 4?s father, two seeminglyclinically unaffected older siblings (no blisters, scars or nail dys-trophy), and the clinically affected child of one of these siblings.Mutation analysis in patient 5, with no family history of EB,revealed compound heterozygosity for a paternally inherited singlebase pair deletion mutation in exon 104, 7786delG, causing a shiftin the reading frame and a premature termination codon in exon106, and a splice site mutation at the 5? donor consensus sequenceat the first nucleotide of intron 64, 5532 ? 1G-to-A. The lattermutation was not identified in parental DNA and had thereforearisen as a de novo event. In patient 6, with autosomal dominantdisease, direct nucleotide sequencing of the exon 87 PCR productrevealed heterozygosity for a 16 bp deletion mutation, 6863del16.This mutation was also present in the patient’s 10 y old daughterwith mild EB pruriginosa features. Other studies have shown thatthis mutation results in in-frame exon skipping of exon 87, whichconsistsof69bp,ratherthanaframeshiftanddownstreamprematuretermination codon (Cserhalmi-Friedman et al, 1998). This phenom-enon has also been described for other autosomal dominant deletionmutations in COL7A1 (Sakuntabhai et al, 1998). Confirmatoryrestriction endonuclease digestions for the mutations in patients1–5, and direct nucleotide sequencing of exon 87 in patient 6, areshown in Fig 1. Apart from the 7786delG mutation present onone allele of patient 5 with recessive EB pruriginosa, none of themutations delineated in this study have been found in other patientswithnonpruriginosavariantsofdystrophicEB.Inaddition,althoughin-frame exon skipping in combination with a premature termina-tioncodonhasbeendescribedinrecessivedystrophicEB(Christianoet al, 1996b), the particular combination of 5532 ? 1G-to-Aand 7786delG has not been reported previously. One possibleconsequence of this splice site mutation could be in-frame exonskipping (Christiano et al, 1996b). Whereas this might be predictedto cause less dominant negative interference when inherited intrans with a premature termination codon rather than a wild-typesecond allele, the splice variant may also compromise type VIIcollagenprocessing, secretion,and anchoringfibril assembly.Unfor-tunately, the precise consequences of this mutation could not bedetermined as a skin biopsy was not available from patient 5. Allthe mutations delineated in this study are illustrated in Fig 2,which also shows all previously described mutations in dominantdystrophic EB.Four of our cases studied had glycine substitution mutations,however, there was no consistency between the position of themutation in the triple helix (exons 61, 85, 93, and 110), nor theresulting amino acid change (glutamic acid, arginine, or serine). Ofinterest, a glycine to aspartic acid substitution has been describedpreviously at amino acid position 2713, G2713D, in nonpruriginosadominant dystrophic EB (Rouan et al, 1998). This is in contrast tothe pruriginosa phenotype in patient 4, in whom a different glycinesubstitution, converting to arginine, was identified at this sameposition. The presence of this mutation in the patient’s father andnephew, with nonpruriginosa features of dominant dystrophic EB,and her phenotypically normal siblings, however, suggests that,whereas the nature of the substituted amino acid may be relevanttotheresultingphenotype,additionalfactorsmayalsobeimplicated.Although intrafamilial variability in dominant dystrophic EB is wellrecognized, it appears to be particularly evident in EB pruriginosa,as demonstrated in the family of patient 4. Clearly, therefore,accurate genetic counseling for this condition must involve screen-ing of genomic DNA of all family members and must not be basedon clinical examination alone, even if careful assessment is madeof subtle signs such as minor nail dystrophy.Figure 2 demonstrates that glycine substitution mutations givingrise to dominant dystrophic EB are distributed across the triplehelix with clustering in exon 73 close to the noncollagenous hingeregion.Nevertheless,someevidenceforclusteringofEBpruriginosamutations in a different part of COL7A1 has been suggested byTamai et al1who identified glycine substitutions in exon 85. Thiswas not corroborated by our series, however.In view of the heterogeneous nature of these mutations, weassessed other indices that might account for the pruritus in ourcases. Serum IgE levels were measured in two of the patientsstudied and in a further seven with EB pruriginosa, and found to

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986MELLERIO ET ALTHE JOURNAL OF INVESTIGATIVE DERMATOLOGYFigurepruriginosa mutations is shown byrestrictionendonuclease(patients 1–5 and control, C) or directnucleotidesequencing(a) In patient 1, SacI digestion of exon 85mismatch PCR products cuts the 207 bpPCR product into 187 bp and 20 bpfragments on the mutant G2242R allele.(b) G1791E creates a BlpI restrictionendonuclease site. In patient 2, the 506 bpPCR product is digested into additionalbands of 381 bp and smaller fragments.(c) In patient 3, G2369S abolishes a HphIrestriction site. Control PCR product iscut into fragments of 197 bp, 55 bp, and59 bp, whereas patient 3 has an additionalundigested 252 bp band. (d) The controlPCRproductspanningexon110isdigestedby Eco01091 into fragments of sizes 104 bp,97 bp, and ? 70 bp. In patient 4, G2713Rabolishes an Eco01091 restriction site,resulting in an additional undigested bandof 128 bp (e) 5532 ? 1G-to-A abolishes aTaiI restriction site. Control exon 63/64PCR product is digested into 247 bp andsmaller fragments, whereas patient 5 has anundigested 312 bp band corresponding tothe mutant allele. (f) 7786delG abolishes aBsaJI cut site. The control 512 bp exon103/104 PCR product is digested into? 60 bp fragments, but patient 5 hasan additional band of 120 bp. (g) Directnucleotide sequencing of exon 87 PCRproducts from patient 6 reveals a 16 bpdeletion, 6863del16, on one allele. Wild-type (top) and mutant (bottom) sequencesare shown from subcloned PCR products.(h) Gel electrophoresis of the 404 bp exon86/87 PCR products shows a second bandof 388 bp in patient 6 corresponding tothe mutant allele (3% Nu-Sieve agarose gel;Flowgen, Litchfield, U.K.). M ? ΦX174molecular weight marker.1. Verification ofthe EBdigestion(patient 6).be elevated in seven cases (112→1000 kU per liter; normal range0–81 kU per liter), and to at least three times the upper limit ofnormal in three of these patients. Of these seven patients, one hada personal history of eczema, and two had family, but not personal,histories of atopy. Serum IgE levels were also found to be elevatedin two of six control patients with nonpruriginosa dystrophic EBwho were not known to be atopic. Clearly, an elevated IgE doesnot specifically account for a pruriginosa phenotype and additionalpotential predisposing factors for atopy, such as alterations in the βsubunit of the high-affinity IgE receptor (Cox et al, 1998) orcytokine polymorphisms (Mitsuyasu et al, 1998) may be implicated.The described clinical improvement of EB pruriginosa with cyclo-sporine A is supportive of additional immune-mediated factors inthe pathogenesis of this disease (Yamasaki et al, 1997). There wasno evidence for other causes of itching in the patients studied, suchas thyroid dysfunction or low ferritin levels. The pathophysiologyofitchispoorlyunderstood,however,andotherpotentialmodifyingfactors remain to be elucidated.The reasons for the frequently delayed onset of clinical featuresin EB pruriginosa remains unclear. The position of mutations inthe type VII collagen triple helix may be significant, with mostglycine substitution mutations in nonpruriginosa dominant dys-trophic EB clustered around exon 73. Mutations in other regionsof the triple helix, as observed in our patients, may cause lessdominant negative interference and only result in clinical diseasewhen combined with additional inherited or acquired predisposingfactors. Similarly, the position of a glycine substitution mutationmay influence the susceptibility of type VII collagen to secondaryproteolysis, with potential for differences in breakdown productstriggering an inflammatory cascade in the patient’s skin.This study demonstrates the molecular heterogeneity underlyingEB pruriginosa, with glycine substitution mutations, a deletion

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[Show abstract][Hide abstract]ABSTRACT:
Epidermolysis bullosa pruriginosa is a rare distinctive variant of dystrophic epidermolysis bullosa characterized by intense pruritus, lichenified plaques in linear distribution, and anonychia. It is a difficult condition to treat and causes a great deal of distress. The present authors report two cases showing good response to low-dose thalidomide, with clinical and symptomatic improvement. The exact mechanism of action is not yet clear.

[Show abstract][Hide abstract]ABSTRACT:
Epidermolysis bullosa pruriginosa is a rare variant of dystrophic epidermolysis bullosa characterized by severely pruritic and cicatricial lesions localized to the extensor extremities. We report a Singaporean Chinese male with epidermolysis bullosa pruriginosa with an underlying novel mutation in the COL7A1 gene. A heterozygous acceptor splice site mutation IVS67-1G>T probably led to in-frame skipping of exon 68 (36-basepairs), resulting in a loss of 12 amino acids. Among his three children, only the youngest son, who had bilateral big toenail thickening, possessed the same mutation. His skin biopsy one decade ago revealed association of focal amyloidosis; a recent skin biopsy showed more established features of lichen amyloidosis. It is debatable whether the cutaneous amyloidosis was a secondary or primary phenomenon. Our report highlights that the diagnosis of epidermolysis bullosa pruriginosa may be obscured when cutaneous amyloidosis is coexistent.